Escherichia coli O157:H7 poses a significant threat to human health and food safety. Traditional agents used for antibacterial control can be hazardous to health and cause environmental pollution, consequently, plant-derived antimicrobials have attracted considerable interest as promising alternatives. In the study, the antimicrobial activity and mechanisms of the terpenoid eucalyptol (1,8-cineole) against E. coli O157:H7 were investigated for the first time, based on transcriptomics and molecular docking analyses. Eucalyptol was demonstrated to inhibit the growth of E. coli O157:H7 with minimum inhibitory and bactericidal concentrations of 1.8 and 3.0 μL/mL, respectively. In addition, results indicated that eucalyptol altered E. coli O157:H7 cell morphology, disrupted cell membrane integrity, and contributed to the leakage of nucleic acids and proteins. Transcriptomic analysis revealed that exposure to eucalyptol interfered with the bacterial quorum sensing system, inhibited flagellar motility and adhesion, and resulted in the differential expression of 38 genes closely associated with bacterial biofilm formation, virulence, and survival. Furthermore, molecular docking results indicated that gadA-encoded glutamate decarboxylase B (GadB) and autoinducer-2 degrading protein (LsrG) in E. coli O157:H7 could represent potential targets for eucalyptol. Hemolysis assays showed that the hemolysis rate below 5 %, demonstrating acceptable in vitro safety. Moreover, the E. coli O157:H7 in beef broth was completely eradicated by 2 × MIC of eucalyptol during storage. These findings revealed the potential of eucalayptol as a new natural food antibacterial agent and preliminarily elucidated the antibacterial mechanism, providing a new strategy for controlling E. coli O157:H7 in the food industry.